INTRODUCTION

Section:

ChooseTop of pageABSTRACTINTRODUCTION <<RESULTSDISCUSSIONCONCLUSIONSMETHODSDiabetes is a chronic degenerative disease that occurs when the pancreas does not produce enough insulin or the body cannot effectively use it. In 2019, approximately 463 million adults aged 20 and 79 were living with diabetes; by 2045, this will rise to 700 million.11. See https://www.diabetesatlas.org for “ IDF Diabetes Atlas, Ninth edition,” 2019 (last accessed September, 2021). This illness has become the seventh leading cause of death in the United States and the leading cause of amputation, blindness, and renal failure.22. Y. Huang and T. R. Kyriakides, “ The role of extracellular matrix in the pathophysiology of diabetic wounds,” Matrix Biol. Plus 6–7, 100037 (2020). https://doi.org/10.1016/j.mbplus.2020.100037 In addition, the cost of wound treatment ranges from 28.1 billion to 96.8 billion US dollars due to the increase in the aging and diabetic population.33. H. Ma, P. K. Lam, W. S. Siu, C. S. W. Tong, K. K. Y. Lo, C. M. Koon, X. X. Wu, X. Li, W. Cheng, W. T. Shum, and P. C. Leung, “ Adipose tissue-derived mesenchymal stem cells (ADMSCs) and ADMSC-derived secretome expedited wound healing in a rodent model: A preliminary study,” Clin. Cosmet. Investig. Dermatol. 14, 753–764 (2021). https://doi.org/10.2147/CCID.S298105According to the American Diabetes Association (ADA), this pathology is a complex chronic disease requiring continuous medical care with multifactorial risk reduction strategies for glycemic control. Moreover, this disease has many medical complications, such as myocardial infarction, cerebrovascular accidents, kidney failure, amputation of lower limbs, and neuropathy.44. American Diabetes Association, “ Standards of medical care in diabetes-2017 abridged for primary care providers,” Clin. Diabetes 35(1), 5–26 (2017). https://doi.org/10.2337/cd16-0067 It has been highlighted that between 30% and 70% of patients with diabetes, both type 1 and type 2, will present a diabetes cutaneous complication at some point during their lifetime, associated with an increased risk of essential outcomes, such as skin lesions, ulcerations, and diabetic foot, which can lead to further significant complications.5,65. J. Rosen and G. Yosipovitch, “ Skin manifestations of diabetes mellitus,” in Endotext (Internet), edited by K. R. Feingold , B. Anawalt , A. Boyce , G. Chrousos , W. W. de Herder , K. Dhatariya , K. Dungan , J. M. Hershman , J. Hofland , S. Kalra , G. Kaltsas , C. Koch , P. Kopp , M. Korbonits , C. S. Kovacs , W. Kuohung , B. Laferrère , M. Levy , E. A. McGee , R. McLachlan , J. E. Morley , M. New , J. Purnell , R. Sahay , F. Singer , M. A. Sperling , C. A. Stratakis , D. L. Trence , and D. P. Wilson ( MDText.com, Inc., South Dartmouth, MA, 2021).6. G. M. de Macedo, S. Nunes, and T. Barreto, “ Skin disorders in diabetes mellitus: An epidemiology and physiopathology review,” Diabetol. Metab. Syndr. 8(1), 63 (2016). https://doi.org/10.1186/s13098-016-0176-y Furthermore, some studies have demonstrated that most diabetic amputations precede foot ulceration, resulting in severe gangrene or infection.77. V. T. Nguyen, N. Farman, R. Palacios-Ramirez, M. Sbeih, F. Behar-Cohen, S. Aractingi, and F. Jaisser, “ Cutaneous wound healing in diabetic mice is improved by topical mineralocorticoid receptor blockade,” J. Invest. Dermatol. 140(1), 223–234 (2020). https://doi.org/10.1016/j.jid.2019.04.030On the other hand, human skin is a physical barrier with multiple functions; it works as an interface between the human body and its environment, prevents water loss, and protects the body from physical, chemical, and biological insults. Moreover, it contributes to the vascular capacity of the entire body, blood pressure control, and glucose handling.88. W. Groenendaal, G. von Basum, K. A. Schmidt, P. A. Hilbers, and N. A. van Riel, “ Quantifying the composition of human skin for glucose sensor development,” J. Diabetes Sci. Technol. 4(5), 1032–1040 (2010). https://doi.org/10.1177/193229681000400502 Nevertheless, some of these functions are disrupted in diabetic people.As known, wound healing is a physiological reaction to tissue injury involving a complex interplay between numerous cell types, cytokines, mediators, and the vascular system. It begins with homeostasis and ends with scar tissue formation; the necrotic tissue is removed by scavenger cells or separated from living tissue by the process of phagocytosis. The wound healing process consists of four phases: (1) Hemostasis: in this phase, vasoconstriction of blood vessels and platelet aggregation is carried out; the clot provides a matrix for the cells involved in the subsequent steps of hemostasis and inflammation. (2) Inflammation: the goal of the inflammatory phase is to fight against possible bacterial contamination and to activate cytokine secretion; the duration of the inflammatory stage usually lasts several days. (3) Proliferation: this is characterized by granulation (dermis repair), re-epithelialization (formation of a new epidermis), and angiogenesis; this phase can last several weeks. (4) Remodeling: once the fibrin clot is formed, it is replaced by granulation tissue rich in collagen types III and I. In this phase, the wound achieves maximum strength as it matures.9–119. A. Gupta and P. Kumar, “ Assessment of the histological state of the healing wound,” Plast. Aesthet. Res. 2, 239–242 (2015). https://doi.org/10.4103/2347-9264.15886210. H. A. Wallace, B. M. Basehore, and P. M. Zito, Wound Healing Phases ( StatPearls Publishing, Treasure Island, FL, 2022).11. J. Benavides, “ Reparación de heridas cutáneas,” Rev. Asoc. Colomb. Dermatol. Cir. Dermatol. 16(1), 29–35 (2008).It is essential to mention that in diabetes, extracellular matrix proteins undergo glycation-induced modification leading to the formation of advanced glycation end products. This is why collagen is subjected to non-enzymatic glycosylation, provoking some people with diabetes to have thickened, waxy skin.2,122. Y. Huang and T. R. Kyriakides, “ The role of extracellular matrix in the pathophysiology of diabetic wounds,” Matrix Biol. Plus 6–7, 100037 (2020). https://doi.org/10.1016/j.mbplus.2020.10003712. N. H. Cox, D. McCruden, A. McQueen, S. K. Jones, L. Ong-Tone, A. Y. Finlay, and B. M. Frier, “ Histological findings in clinically normal skin of patients with insulin-dependent diabetes,” Clin. Exp. Dermatol. 12(4), 250–255 (1987). https://doi.org/10.1111/j.1365-2230.1987.tb01913.xIn addition, more than 100 physiological factors are involved in the healing process, and some do not work well in people with diabetes, entailing poor wound healing. These include decreased production of growth factors, alterations in angiogenic response, macrophage function, collagen accumulation, epidermal barrier function, amount of granulation tissue, migration and proliferation of keratinocytes and fibroblasts, number of epidermal nerves, healing bone, and imbalance between the accumulation of the extracellular matrix (ECM) components and their remodeling by matrix metalloproteinases (MMPs).1313. C. Qing, “ The molecular biology in wound healing & non-healing wound,” Chin. J. Traumatol. 20(4), 189–193 (2017). https://doi.org/10.1016/j.cjtee.2017.06.001Current treatments for wound healing include growth factors and cytokines, skin substitutes, hyperbaric oxygen therapy, and skin grafts. However, these conventional treatments are not effective, probably due to the complex mechanisms involved in developing unhealed wounds, which is why regenerative medicine using stem cells could be a novel tool for treating non-healing ulcers.33. H. Ma, P. K. Lam, W. S. Siu, C. S. W. Tong, K. K. Y. Lo, C. M. Koon, X. X. Wu, X. Li, W. Cheng, W. T. Shum, and P. C. Leung, “ Adipose tissue-derived mesenchymal stem cells (ADMSCs) and ADMSC-derived secretome expedited wound healing in a rodent model: A preliminary study,” Clin. Cosmet. Investig. Dermatol. 14, 753–764 (2021). https://doi.org/10.2147/CCID.S298105

Therefore, it is essential to analyze the healing process biomolecularly, using precise, fast, and less expensive methods, and to investigate and propose effective treatments that improve healing or increase skin regeneration.

In this sense, Fourier transform infrared (FTIR) spectroscopy is a valuable tool in studying biological samples since it provides information on the molecular structure of organic and inorganic materials. In this technique, the absorption of infrared radiation (IR) occurs when a photon is transferred to a molecule and excites it to a higher energy state, giving rise to vibrations of molecular bonds, which occur at different wave sizes or frequencies in the IR region of the light spectrum.1414. M. M. Mata-Miranda, M. Guerrero-Ruiz, J. R. Gonzalez-Fuentes, C. M. Hernandez-Toscano, J. R. Garcia-Andino, M. Sanchez-Brito, and G. J. Vazquez-Zapien, “ Characterization of the biological fingerprint and identification of associated parameters in stress fractures by FTIR spectroscopy,” Biomed. Res. Int. 2019, 1241452. https://doi.org/10.1155/2019/1241452Different biomolecules, such as lipids, proteins, carbohydrates, and nucleic acids, can be detected in biological materials with specific chemical structures. In addition, by coupling FTIR spectroscopy with optical microscopy, analysis using FTIR microspectroscopy (FTIRM) has been possible, which allows the visualization and mapping of functional groups and molecular arrangements.1515. G. J. Vazquez-Zapien, M. M. Mata-Miranda, F. J. Garcia-Sanchez, S. S. Campos-Soto, M. Sanchez-Brito, and A. Martinez-Cuazitl, “ Biomolecular characterization by FTIR microspectroscopy in the modeling phase of wound cicatrization in a murine model of excisional injury,” Int. J. Morphol. 4, 1234–1244 (2019). https://doi.org/10.4067/S0717-95022019000401234 This method has been called “chemical photography,” as semi-quantitative information can be obtained from IR spectra using standard image processing software.1616. F. Palombo, S. G. Cremers, P. D. Weinberg, and S. G. Kazarian, “ Application of Fourier transform infrared spectroscopic imaging to the study of effects of age and dietary L-arginine on aortic lesion composition in cholesterol-fed rabbits,” J. R. Soc. Interface 6(37), 669–680 (2009). https://doi.org/10.1098/rsif.2008.0325The FTIRM is sensitive to determining the distribution and orientation of various components, which has been used to recognize multiple pathologies; this technique makes it possible to detect concentrations of molecules using the area under the curve of specific peaks in the spectrum as well as the radii between particular areas.1515. G. J. Vazquez-Zapien, M. M. Mata-Miranda, F. J. Garcia-Sanchez, S. S. Campos-Soto, M. Sanchez-Brito, and A. Martinez-Cuazitl, “ Biomolecular characterization by FTIR microspectroscopy in the modeling phase of wound cicatrization in a murine model of excisional injury,” Int. J. Morphol. 4, 1234–1244 (2019). https://doi.org/10.4067/S0717-95022019000401234 For example, it is possible to analyze the secondary structure of proteins employing the second derivative or deconvolution, examining bands corresponding to α-helices, β sheets, β turns, and disordered structure.1717. B. Balázs, G. Farkas, O. Berkesi, R. Gyulai, S. Berkó, M. Budai-Szucs, P. Szabó-Révész, L. Kemény, and E. Csányi, “ Protein structure is changed in psoriatic skin on the unaffected region—Imaging possibility with ATR-FTIR spectroscopy,” Microchem. J. 117, 183–186 (2014). https://doi.org/10.1016/j.microc.2014.07.003 The specificity and advantages of using the second derivative have made it possible to determine the number and position of peaks of interest, especially in the region of amide I proteins,1818. E. P. Paschalis, S. Gamsjaeger, D. N. Tatakis, N. Hassler, S. P. Robins, and K. Klaushofer, “ Fourier transform infrared spectroscopic characterization of mineralizing type I collagen enzymatic trivalent cross-links,” Calcif. Tissue Int. 96(1), 18–29 (2015). https://doi.org/10.1007/s00223-014-9933-9 and hence, its usefulness in wound regeneration could reveal molecular data not described far.

In this sense, regenerative medicine has focused on diabetes, not only in the search to decrease glucose levels but also in the pursuit to improve the healing process due to the increased risk of developing skin lesions in diabetic people. Therefore, due to the poor biomolecular understanding of diabetic skin and the effects of treating it with stem cells, in this work, we analyzed in a nondestructive and specific way an experimental treatment of PSCs in a post-skin lesion excision diabetic murine model, correlating the morphometric, histological, and microspectroscopic characteristics of the wound.

RESULTS

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ChooseTop of pageABSTRACTINTRODUCTIONRESULTS <<DISCUSSIONCONCLUSIONSMETHODS

Murine diabetic model

As previously mentioned, the capillary blood glucose was monitored. Before diabetes induction, capillary glucose was evaluated in both groups. The ISS group exhibited 133.70 ± 31.68 mg/dl, and the PSCs group exhibited 126.60 ± 32.37 mg/dl. Three days after diabetes induction, both groups showed higher glucose levels; the ISS group presented 515.40 ± 68.97 mg/dl, and the PSCs group presented 516.00 ± 63.06 mg/dl; no statistical significance was observed between the groups along the skin healing process. On day 21, the ISS group presented 586.40 ± 24.12 mg/dl, and the PSCs group presented 576.30 ± 29.44 mg/dl (Fig. 1), highlighting that no treatment was given for the diabetes control.

Morphometric analysis of the excisional lesion

Morphological analysis was performed by a photograph record obtained when the injury was made (day 0) and then on days 2, 4, 6, 8, 10, 12, and 15. In Fig. 2(a), representative photos of one mouse of each group are shown; the wound healing process over time can be noticed, highlighting that in the ISS group, no hair is evidenced, which is in contrast with the PSCs group where the hair surrounds the wound; even more, some hairs are shown in the scab.In Fig. 2(b), the means of the lesion areas are tabulated. On day 0, it can be observed that the skin lesion excisions in both groups are pretty similar; the ISS group presented an area lesion of 99.46 ± 4.07 mm2, and the PSCs group presented an area lesion of 99.99 ± 4.71 mm2. Two days post-skin lesion excision in both groups, the area lesion was reduced; the ISS presented an area lesion of 85.44 ± 3.17 mm2, and the PSCs group presented an area lesion of 86.46 ± 3.76 mm2. Both groups showed a decrease in the lesion area throughout the healing time. However, no statistical significance was observed between the groups. On day 15 post-skin lesion excision, the ISS group presented an area lesion of 39.62 ± 1.73 mm2 and the PSCs group presented an area lesion of 39.10 ± 1.25 mm2. Figure 2(c) shows the percentage of skin lesions over time.

Histological analysis

The histopathological samples of both groups (ISS and PSCs) were stained employing hematoxylin & eosin and were evaluated at 7 and 15 days post-skin lesion excision. In Fig. 3, representative microphotographs are shown; along the wound healing process, different stages were identified, including coagulation, granulation tissue formation, re-epithelialization, and ECM remodeling. On day 7 post-skin lesion excision in the ISS group, a hematic scab (1) that lines the wound bed can be evidenced and the subcostraceous epithelialization (2) caused by proteolytic enzymes that make their way under the fibrin crust (from which they can get fed). In addition, a separation of the papillary dermis (3) due to edema and migration of inflammatory cells was observed, and finally, the lack of skin annexes (hair follicles, sebaceous, and sweat glands) was observed.

In contrast, in the PSCs group, the scab is less solid, and the subcostraceous epithelialization process (4) is not as evidenced as in the ISS group, highlighting the separation of the papillary dermis was observed in the ISS was not observed in the PSCs group. Moreover, the collagen fibers in the dermis were examined, noticing a higher collagen fiber density in the PSCs group than in the ISS (these are observed in pink). Furthermore, it is essential to mention that, as seen in the macroscopic examination, skin annexes were observed in the dermis of the PSCs group.

On day 15, post-skin lesion excision, in the macroscopic analysis through the photograph record, it can be observed that the scab remains; nevertheless, due to the handling of the sample, it was lost in the process of the histological technic. However, an epithelialization below the covering surface where the scab should have been placed was observed; it was also evidenced that the epidermis thickened with a more significant number of keratinocytes, persisting a slight sign of edema in the dermis layer. However, a demarcation line comprised mainly of polymorphonuclear leukocytes (PMN) and granulation tissue (5) is observed. When comparing the healing process in the ISS group on day 7 post-skin lesion excision, the better polarity of the keratinocytes and a greater density of collagen fibers were observed. In contrast, the PSCs group on day 15 exhibited a greater amount of granulation tissue (5), as well as collagen fibers, highlighting the presence of skin annexes [hair follicles (6)] (Fig. 3).Figure 4 shows the histopathological analysis at 7 and 15 days post-skin lesion excision of both groups (ISS and PSCs) through picrosirius red stain. The use of light microscopy allows the distinction of collagen fibers, which are colored in red. On the other hand, polarized light microscopy produces a birefringence, provoking that type I collagen fibers (thick fibers) to be seen in yellow-orange and type III collagen fibers (thin fibers) to be seen in green. Therefore, the emitted luminescence was evaluated to determine the content of the collagen fibers. As seen, type I collagen fibers are abundant in healthy skin (171 141.34 ± 27.43 lm), but they considerably decreased at day 7 post-skin lesion excision in the ISS (42 683.7 ± 26.87 lm) and in the PSCs groups (115 126.55 ± 14.92 lm).Moreover, at day 15 post-skin lesion excision, these fibers showed an increment in the ISS group (115 719.11 ± 16.4 lm) and PSCs group (130 955 ± 15.54 lm). In the same way, the type III collagen fibers were abundant in the healthy skin (194 439.25 ± 30.02 lm) and decreased at day 7 post-skin lesion excision in the ISS group (36 123.76 ± 27.87 lm) and the PSCs group (76 745.96 ± 14.25 lm); the experimental groups showed a higher content of type III collagen fibers, but they slightly recovered at day 15 post-skin lesion excision in the ISS group (92 989.32 ± 17.5 lm) and also in the PSCs groups (87 111.29 ± 11.67 lm). However, the type III collagen fibers are more abundant in the PSCs group compared to the ISS group (Fig. 5).The epidermis thickness was also evaluated; Fig. 6 depicted the epidermis thickness of healthy mice skin and scarred treated with ISS or PSCs, according to their group. As observed, the thickness of the epidermis increased throughout the healing time. For example, the thickness of a healthy epidermis was 28.87 ± 10.81 μm; nevertheless, the epidermis of the ISS group healed on days 7 and 15 post-skin lesion excision measured 78.63 ± 18.55 and 99.87 ± 20.84 μm, respectively. In the same way, the epidermis of the PSCs group healed at 7 and 15 days post-skin lesion excision also showed an increment in the epidermis thickness, measuring 68.38 ± 19.69 and 101.2 ± 17.48 μm, respectively, observing that even though no statistical significance was found, the ISS group presented a thicker epidermis that the PSCs group at day 7 post-skin lesion excision. Nevertheless, on day 15, the PSCs group exhibited a thicker epidermis than the ISS group.As previously mentioned, cellularity was also evaluated, for which purpose the cellularity of healthy mice skin dermis was analyzed and compared to scarred dermis treated with ISS or PSCs at days 7 and 15 post-skin lesion excision. Figure 7 shows that both groups' cellularity increased at day 7 post-skin lesion excision. However, the PSCs group showed less cellularity than the ISS group. Moreover, on day 15 post-skin lesion excision, the cellularity diminished, but it remained increased compared to healthy skin in the ISS group. Nonetheless, the PSCs group exhibited less cellularity than the healthy skin, showing statistical significance.

FTIR microspectroscopy

Regarding the spectroscopic results using FTIRM, Fig. 8 shows the normalized and averaged FTIR spectra of the epidermis and dermis of healthy skin, as well as skin biopsies obtained on days 7 and 15 post-skin lesion excision of ISS and PSCs groups, where the absorption bands related to lipids (1737 and 1456 cm−1), proteins [amide I (1666 cm−1) and amide II (1549 cm−1)], collagen (1400 cm−1), phospholipids (1246 cm−1), and nucleic acids (1085 cm−1) are depicted.

It can be observed that the band associated with lipids showed a higher absorbance in the PSCs group compared to the ISS group at the different analyzed times. Moreover, the bands related to amides I and II exhibited a higher absorbance in the dermis section of both groups.

Furthermore, the band associated with phospholipids showed a higher absorbance in the epidermis and dermis sections on day 7 than the ISS group; nevertheless, on day 15 in the epidermis sections, the phospholipids band was higher in the ISS group. However, this band remains a higher absorbance in the PSCs group in the dermis section. Finally, the band related to nucleic acids showed a higher absorbance in the epidermis section of the PSCs group on day 7; however, on day 15, the ISS and the PSCs group presented similar absorbance. Moreover, in the dermis section, the nucleic acid band exhibited a slight increase in absorbance in the ISS group; nevertheless, on day 15, the PSCs group presented a higher absorbance.

On the other hand, to specifically analyze the secondary structure of proteins, the second derivative of normalized FTIR spectra of the epidermis and dermis of healthy skin as well as skin biopsies obtained on days 7 and 15 post-skin lesion excision of ISS and PSCs groups in the amide I region of proteins (1700–1600 cm−1) was calculated (Fig. 9), where the following bands are observed: β sheets at 1685 cm−1, α-helices at 1651 cm−1 corresponding to the keratin of the epidermis, and at 1660 cm−1 to collagen dermis. Moreover, the disordered structure of proteins at 1638 cm−1 was also detected. In the epidermis sections, it can be observed that the band attributed to β sheets showed a higher absorbance in the ISS groups on days 7 and 15; in contrast, the band associated with keratin exhibited a higher absorbance in the PSCs group on days 7 and 15. Finally, in the epidermis section, the band related to the disordered structure of proteins showed a higher absorbance in the PSCs group; nevertheless, on day 15, the ISS group exhibited a higher absorbance. In the same way, in the dermis sections, the band attributed to β sheets at 1685 cm−1 showed a higher absorbance in the ISS group on day 7; nevertheless, on day 15, the absorbance decreased. The band associated with collagen at 1660 cm−1 showed a higher absorbance in the PSCs group on day 7; however, on day 15, the ISS group presented a higher absorbance. Regarding the disordered structure of the proteins at 1638 cm−1, this band showed a higher absorbance in the PSCs group at day 7 in the epidermis and dermis section than in the ISS group. Nevertheless, in both sections, this band evidenced a higher absorbance in the ISS group than in the PSCs group on day 15.

Mapping analysis

The histological localization and concentration of some crucial molecules in skin bioconformation, including lipids, ceramides C=O amide I, ceramides N–H/C–N amide II, collagen, collagen triple helix, and β-sheet structure content was developed using the IQ mapping function. Figure 10 shows a representative image of the mapping analysis. The microscope image illustrates the skin biopsy in which the analysis was carried out; in the same way, the total absorbance images from FTIRI are presented. Each image represents the integrated absorbance of a specific band of the IR spectra for each pixel of the MCT detector; the red and blue colors represent the strong and weak absorption of the infrared beam.

According to analysis mentioned above, the PSCs group on days 7 and 15 post-skin lesion excision showed a higher lipid content than the ISS group. Nevertheless, ceramides of amide I and amide II presented a greater content in the ISS group on day 7, which significantly decreased on day 15; however, in the PSCs group, the ceramide content remained expressed in a very subtle way. The analysis of the collagen content showed that this molecule showed a higher absorbance on day 7 in both groups, but on day 15, this molecule decreased considerably in the ISS group. The collagen fibers aligned were analyzed through the amide I/amide II ratio, whereas the previously mentioned higher values represent an unordered collagen fiber structure with multiple directions; on day 7, both groups showed almost the same organization of collagen fibers. Nevertheless, on day 15, it can be noticed that the ISS group exhibited an unordered collagen fiber structure, which is in contrast with the PSCs group, which showed ordered fibers. Furthermore, the collagen triple helix analysis evidenced that the PSCs group on days 7 and 15 showed a significant content of this molecule; as seen in the collagen content, the collagen triple helix in the ISS group decreased considerably on day 15. Finally, the disordered structure of proteins and collagen fibers aligned exhibited a great content in the ISS group on days 7 and 15 post-skin lesion excision compared to the PSC group. It is important to mention that all the mapping analysis of the PSCs group at day 15 were quite similar to those presented in healthy skin.

Biochemical content

The areas under the curve of the bands related to lipids, ceramides C=O amide I, ceramides N–H/C–N amide II, collagen, collagen fibers aligned, collagen triple helix, and α–β transition were calculated in both groups on days 7 and 15 post-skin lesion excision as well as in healthy skin (Fig. 11). It is noticed that lipids, ceramides, and collagen decreased in both groups (ISS and PSCs) with respect to the healthy skin. Regarding lipid content, this molecule increased over time; moreover, the PSCs group in the dermis section showed a more significant amount than the ISS group on days 7 and 15 post-skin lesion excision; nevertheless, the epidermis section did not show differences in content. About the content of ceramide C=O amide I and ceramides N–H/C–N amide II, these molecules decreased in the epidermis section on day 15 post-skin lesion excision compared to day 7. In contrast, it slightly increased on day 15 in the dermis section; however, it is essential to mention that the dermis section presented greater biomolecule content. Regarding collagen amount, its content was more significant in the epidermis sections on days 7 and 15 post-skin lesion excision, but when compared between groups, as seen in the mapping analysis, the PSCs group showed more collagen in the epidermis and dermis sections on day 7 and 15. About the collagen fibers aligned, analyzed with the amide I/amide II ratio, no statistical differences were evidenced on day 7 between groups; nevertheless, on day 15, the ISS group showed a more significant amount of this biomolecule. The analysis of the collagen triple helix showed that the epidermis region has more content of this molecule at days 7 and 15 post-skin lesion excision than the ISS group. Finally, the content of α–β transition was higher in the PSCs group than in the ISS group on days 7 and 15 post-skin lesion excision in the epidermis and dermis sections, highlighting that a decrement was observed on day 15 in the epidermis section of the ISS group. Moreover, on day 15 in the dermis section, an increasement was evidenced.

DISCUSSION

Section:

ChooseTop of pageABSTRACTINTRODUCTIONRESULTSDISCUSSION <<CONCLUSIONSMETHODS

This experimental study was designed to examine the impact of PSCs as a treatment in the cicatrization process in a diabetes skin lesion, for the purpose of which an excisional skin model in diabetic mice was developed. The lesions were treated with PSCs and ISS according to the group that the animals belonged to, and different characteristics of the wound, such as morphometric, histological, and microspectroscopic characteristics, were studied on days 7 and 15 post-skin lesion excision.

In this research, we decided to use male mice as it has been reported that the male skin is 40% stronger due to a much thicker dermis, while female skin exhibits a thicker epidermis.2121. D. S. Masson-Meyers, T. A. M. Andrade, G. F. Caetano, F. R. Guimaraes, M. N. Leite, S. N. Leite, and M. A. C. Frade, “ Experimental models and methods for cutaneous wound healing assessment,” Int. J. Exp. Pathol. 101(1–2), 21–37 (2020). https://doi.org/10.1111/iep.12346 Moreover, some studies have concluded that in females mice, estrogens such as 17β-estradiol accelerate the wound healing process.2222. S. C. Gilliver, E. Emmerson, L. Campbell, P. Chambon, M. J. Hardman, and G. S. Ashcroft, “ 17β-estradiol inhibits wound healing in male mice via estrogen receptor-alpha,” Am. J. Pathol. 176(6), 2707–2721 (2010). https://doi.org/10.2353/ajpath.2010.090432For the development of diabetic mice in this research, we used STZ, which many authors have used to induce diabetes in animal models. King has stated that STZ is one of the leading compounds to chemically cause models of type 1 diabetes, provoking a high percentage of destruction of endogenous beta cells. Moreover, after i.p. administration, it enters the pancreatic beta cell through the Glut-2 transporter, provoking the alkylation of the DNA and leading to hyperglycemia. King has declared that mice's ideal single high dose ranges from 100 to 200 mg/kg.2323. A. J. King, “ The use of animal models in diabetes research,” Br. J. Pharmacol. 166(3), 877–894 (2012). https://doi.org/10.1111/j.1476-5381.2012.01911.x The dose employed in this research was 175 mg/kg, with which stable high glycemic values around 500 mg/dl were reached, and it remained high until the end of the experiment, 586.40 ± 24.12 mg/dl for the ISS group and 576.30 ± 29.44 mg/dl for the PSCs group.Regarding the excisional lesion model, Ma et al. investigated the therapeutic effects of mesenchymal stem cells (MSCs) and secretome in the excisional wound model, making a full-thickness skin excision of 2.5 × 2.5 cm2 on the dorsal area of adult female SD rats.33. H. Ma, P. K. Lam, W. S. Siu, C. S. W. Tong, K. K. Y. Lo, C. M. Koon, X. X. Wu, X. Li, W. Cheng, W. T. Shum, and P. C. Leung, “ Adipose tissue-derived mesenchymal stem cells (ADMSCs) and ADMSC-derived secretome expedited wound healing in a rodent model: A preliminary study,” Clin. Cosmet. Investig. Dermatol. 14, 753–764 (2021). https://doi.org/10.2147/CCID.S298105 Nguyen et al. evaluated the topical mineralocorticoid receptor blockade in cutaneous wound healing in diabetic mice using female mice, in which they generated a 6-mm biopsy punch.77. V. T. Nguyen, N. Farman, R. Palacios-Ramirez, M. Sbeih, F. Behar-Cohen, S. Aractingi, and F. Jaisser, “ Cutaneous wound healing in diabetic mice is improved by topical mineralocorticoid receptor blockade,” J. Invest. Dermatol. 140(1), 223–234 (2020). https://doi.org/10.1016/j.jid.2019.04.030 Moreover, considering that the wound size should be proportional to the animal size and that female mouse skin exhibits a thicker epidermis and subcutaneous layer,2121. D. S. Masson-Meyers, T. A. M. Andrade, G. F. Caetano, F. R. Guimaraes, M. N. Leite, S. N. Leite, and M. A. C. Frade, “ Experimental models and methods for cutaneous wound healing assessment,” Int. J. Exp. Pathol. 101(1–2), 21–37 (2020). https://doi.org/10.1111/iep.12346 in this research, we made an excision of 1 × 1 cm2 on the dorsal area of adult male diabetic mice.About the wound healing process, unlike Ma et al., who reported that the wound healing process was completed after 28 days,33. H. Ma, P. K. Lam, W. S. Siu, C. S. W. Tong, K. K. Y. Lo, C. M. Koon, X. X. Wu, X. Li, W. Cheng, W. T. Shum, and P. C. Leung, “ Adipose tissue-derived mesenchymal stem cells (ADMSCs) and ADMSC-derived secretome expedited wound healing in a rodent model: A preliminary study,” Clin. Cosmet. Investig. Dermatol. 14, 753–764 (2021). https://doi.org/10.2147/CCID.S298105 in this research, the mice were carried out up to 15 days post-skin lesion excision, noticing that they did not reach a complete healing process. Moreover, in contrast to our results, where no statistical significance was reported in the percentage of skin lesions along the time of the wound healing process between the ISS and PSCs groups, Ma et al. reported statistical significance in the healing rate between the control group and the group treated with MSCs. However, on day 14, Ma et al. reported that the healing rate of the MSCs group was around 60%, which was quite similar to the results obtained in this research, where on day 15, the percentage of skin lesions was around 40% in both groups. In addition, it is essential to mention that two days post-skin lesion excision, the injury considerably diminished from 99.46 ± 4.07 and 99.99 ± 4.71 mm2 in the ISS and PSCs groups, respectively, to 85.44 ± 3.34 and 86.64 ± 3.96 mm2, probably due to rodents' skin that has a panniculus carnosus layer, which produces rapid wound contraction following injury.2121. D. S. Masson-Meyers, T. A. M. Andrade, G. F. Caetano, F. R. Guimaraes, M. N. Leite, S. N. Leite, and M. A. C. Frade, “ Experimental models and methods for cutaneous wound healing assessment,” Int. J. Exp. Pathol. 101(1–2), 21–37 (2020). https://doi.org/10.1111/iep.12346As previously mentioned, the wound healing process consists of four phases: (1) hemostasis, (2) inflammation, (3) proliferation, and (4) remodeling.9–119. A. Gupta and P. Kumar, “ Assessment of the histological state of the healing wound,” Plast. Aesthet. Res. 2, 239–242 (2015). https://doi.org/10.4103/2347-9264.15886210. H. A. Wallace, B. M. Basehore, and P. M. Zito, Wound Healing Phases ( StatPearls Publishing, Treasure Island, FL, 2022).11. J. Benavides, “ Reparación de heridas cutáneas,” Rev. Asoc. Colomb. Dermatol. Cir. Dermatol. 16(1), 29–35 (2008). According to this, in the histological analysis of this research, we could observe at least three phases of the wound healing process; however, critical histological differences were detected between groups. For example, on day 7 post-skin lesion excision in the ISS group, a hematic scab evidenced the hemostasis phase and the inflammation phase by separating the papillary dermis and migration of inflammatory cells. In addition, some disorganized collagen fibers were observed. In contrast, the hemostasis phase was characterized by a less solid scab in the PSCs group. However, it is essential to mention that the inflammatory response was regulated once a separation of the papillary dermis was not observed in the inflammatory phase, even though the migration of inflammatory cells was evidenced.Moreover, organized collagen fibers were observed, and the results that agree with Gal et al. (2008), who established in diabetic rats an excisional skin wound healing model employing corticosteroid as treatment. In this research, by day six after the surgery lesion, they found a new layer of epithelial cells in the control group, lightly infiltrated with PMN, and a newly created granulation tissue at the bottom of the wounds.2424. P. Gal, R. Kilik, M. Mokry, B. Vidinsky, T. Vasilenko, S. Mozes, N. Bobrov, Z. Tomori, J. Bober, and L. Lenhardt, “ Simple method of open skin wound healing model in corticosteroid-treated and diabetic rats: Standardization of semi-quantitative and quantitative histological assessments,” Vet. Med. 53, 652–659 (2008). https://doi.org/10.17221/1973-VETMEDOn day 15 post-skin lesion excision (end of proliferation phase) in the ISS group, the scab remained persisting a slight edema sign in the dermis layer with a demarcation line comprised of inflammatory cells, which means that the inflammatory phase was in its final phase. However, granulation tissue, including a smaller amount of collagen, was observed, and an epithelialization was below the covering surface where the scab was placed. In contrast, the PSCs group exhibited a thinner epidermal layer than the ISS group; granulation tissue and new vessels were also evidenced. The proliferation and migration of fibroblasts were decelerated, and the amount of collagen increased with a good deposition and orientation, highlighting the presence of hair follicles. This was partially consistent with Gal et al., who reported that the epidermis regeneration was finished on day 14 of post-surgery in the control group. In addition, the number of fibroblasts and endothelial cells decreased, and the amount of collagen increased simultaneously, forming newly organized fibrils.2424. P. Gal, R. Kilik, M. Mokry, B. Vidinsky, T. Vasilenko, S. Mozes, N. Bobrov, Z. Tomori, J. Bober, and L. Lenhardt, “ Simple method of open skin wound healing model in corticosteroid-treated and diabetic rats: Standardization of semi-quantitative and quantitative histological assessments,” Vet. Med. 53, 652–659 (2008). https://doi.org/10.17221/1973-VETMEDAs previously mentioned, the ISS (control group) did not exhibit hair follicles, which has also been reported by Nguyen et al., who developed a mouse model of diabetes employing STZ, generating wounds of 6-mm by biopsy punch, declaring that no anagen hair follicles were evidenced in the wound healing process, stating that some factors may influence diabetic wound healing, such as abnormalities in hair follicles resulting in defective hair development and cycling, thereby representing a sign of vascular impairment and organ damage in diabetic patients.77. V. T. Nguyen, N. Farman, R. Palacios-Ramirez, M. Sbeih, F. Behar-Cohen, S. Aractingi, and F. Jaisser, “ Cutaneous wound healing in diabetic mice is improved by topical mineralocorticoid receptor blockade,” J. Invest. Dermatol. 140(1), 223–234 (2020). https://doi.org/10.1016/j.jid.2019.04.030The amount of granulation tissue, early collagen, inflammatory infiltrate, vertical orientation of reticular collagen, and minimum amount of mature collagen are the symbol of delayed healing.2525. J. Sultana, M. R. Molla, M. Kamal, M. Shahidullah, F. Begum, and M. A. Bashar, “ Histological differences in wound healing in maxillofacial region in patients with or without risk factors,” Bangladesh J. Pathol. 24(1), 3–8 (2009). https://doi.org/10.3329/bjpath.v24i1.2874 In addition, it is essential to mention that collagen type III is secreted at the early stages of the remodeling phase; it appears between 48 and 72 h and is maximum between five and seven days. After a year or more, the dermis gradually returns to its preinjury phenotype, with a predominance of collagen type I.1111. J. Benavides, “ Reparación de heridas cutáneas,” Rev. Asoc. Colomb. Dermatol. Cir. Dermatol. 16(1), 29–35 (2008). The histopathological analysis through picrosirius red stain allowed us to analyze the remodeling phase in the granulation tissue, evidencing that type III collagen fibers are more abundant in the PSCs group compared to the ISS group. Furthermore, the content of these fibers increased over time, which means that the PSCs group was crossing in the remodeling phase since day 7 post-skin lesion excision, while the ISS group reached this phase until day 15 post-skin lesion excision.

Considering the aforementioned analysis, the general histologic picture of healing in the PSCs group was enhanced compared to the ISS group.

Among the main sources of cells that might be used for wound healing and regeneration of injured skin are embryonic stem cells, induced pluripotent stem cells, and adult stem cells. The reported results using embryonic stem cells or PSCs evidenced epidermis, hair follicles, sebaceous gland regeneration, reduced scar widths, and the promotion of collagen maturity.2626. A. Nourian Dehkordi, F. Mirahmadi Babaheydari, M. Chehelgerdi, and S. Raeisi Dehkordi, “ Skin tissue engineering: Wound healing based on stem-cell-based therapeutic strategies,” Stem Cell Res. Ther. 10, 111 (2019). https://doi.org/10.1186/s13287-019-1212-2 Indeed, some studies have already reported the use of stem cells in wound healing in diabetes, such as Barcelos et al., who evaluated the healing potential of the human fetal aorta-derived CD133+ progenitor cells in a model of ischemic diabetic ulcer, reporting an accelerated wound closure.2727. L. S. Barcelos, C. Duplaa, N. Kränkel, G. Graiani, G. Invernici, R. Katare, M. Siragusa, M. Meloni, I. Campesi, M. Monica, A. Simm, P. Campagnolo, G. Mangialardi, L. Stevanato, G. Alessandri, C. Emanueli, and P. Madeddu, “ Human CD133+ progenitor cells promote the healing of diabetic ischemic ulcers by paracrine stimulation of angiogenesis and activation of Wnt signaling,” Circ. Res. 104(9), 1095–1102 (2009). https://doi.org/10.1161/CIRCRESAHA.108.192138Furthermore, as mentioned in the Results sections, the epidermis thickness was evaluated. As observed, the thickness of the epidermis increased throughout the healing time, indicating that even though no statistical significance was found, the ISS group presented a thicker epidermis than the PSCs group at day 7 post-skin lesion excision. However, on day 15, the PSCs group exhibited a thicker epidermis than the ISS group. Regarding this, it is known that the thickness of the epidermis depends on re-epithelialization events, which are controlled and accelerated by the regulation of inflammatory molecules induced by stem cells.2828. I. Pastar, O. Stojadinovic, N. C. Yin, H. Ramirez, A. G. Nusbaum, A. Sawaya, S. B. Patel, L. Khalid, R. R. Isseroff, and M. Tomic-Canic, “ Epithelialization in wound healing: A comprehensive review,” Adv. Wound Care 3(7), 445–464 (2014). https://doi.org/10.1089/wound.2013.0473 Xiao et al. have mentioned that some studies that have evaluated the use of stem cells in wound healing have been shown to promote the re-epithelialization of skin wounds by inducing the proliferation and differentiation of keratinocytes. In addition, they also reported a decreased inflammatory cell and proinflammatory cytokines, which accelerates the wound healing process by regulating the size of the thickness of the epidermis, generating a controlled process.2929. T. Xiao, Z. Yan, S. Xiao, and Y. Xia, “ P